Impaired proliferation and survival of MST1-deficient T cells in response to activation signals. (A) Control (Ctr.) and MST1-deficient (F2P2) CD4 and CD8 T cells were analyzed in terms of the rate of division (with CFSE dilution indicating cell proliferation) every 24 hours from day 1-3 after PMA/ionomycin–induced activation. Data are representative of 4 independent experiments. (B) Cell death, as analyzed by annexin V binding to control (Ctr.) and MST1-deficient (F2P2) CD4 and CD8 T cells, at different time points (every 24 hours) after CFSE labeling and PMA/ionomycin activation. Data are representative of 2 independent experiments. (C) Absolute cell counts for annexin V⁺ T cells in the CFSE⁺ population presented in panel B. Data are representative of 3 independent experiments. (D) CFSE and annexin V staining of PMA/ionomycin-activated control (Ctr.) and MST1-deficient (F2P2) CD4 and CD8 T cells. Cells were stained with annexin V and sized at different time points by flow cytometry. Histograms depict the CFSE dilution of viable CD4 and CD8 T cells (annexin V⁻). It is important to note that the CFSE graphs are normalized against the number of cells; the histograms represent thepercentage of the maximum signal (% of Max) and do not reflect the number of dividing cells. Data are representative of 2 independent experiments. (E) Cell proliferation, as measured by EdU incorporation in control (Ctr.) and MST1-deficient (F1P1) CD4 and CD8 T cells after 8 days of culture. Cells were labeled with EdU for 60 minutes before fixation. The EdU intensity is shown on the logarithmic y-axis and DNA content (propidium iodide staining) is shown on the linear x-axis. Gates defined the percentage of cells in the G₁, S (EdU positive), and G₂ phases, as presented in the inset. Data are representative of 3 independent experiments. (F) Quantification of EdU incorporation by CD4 and CD8 obtained from control (Ctr.) and MST1-deficient patients (F1P1). Data are representative of 3 independent experiments and are shown as means ± SEM. ***P < .001.